Project Summary/Abstract Immunomodulation through Nanocapsule-Mediated Cytosolic Delivery of siRNA RNA interference is a potentially powerful strategy for immunotherapy. A key barrier to this approach is the inability to effectively deliver siRNA to the cytosol: with current strategies the vast majority of siRNA remains trapped in endosomes and is ineffective. Nanoparticle-stabilized capsules (NPSCs) deliver siRNA directly to the cytosol in a membrane fusion-like process, bypassing endocytosis. We have demonstrated effective knockdown both in vitro and in vivo in the spleen, with the latter requiring significantly lower dosing than current delivery strategies. In our proposed research we will use in vitro and in vivo experiments to optimize the immunomodulatory properties of these vehicles, focusing on reducing inflammatory response by targeting the cytokine TNF-?. Our proposed program features two Aims: Aim 1: We will fabricate and optimize therapeutic siRNA-based NPSCs, focusing on maximizing cytosolar delivery efficiency, carrier capacity, and TNF-? knockdown to macrophages while minimizing toxicity and non-specific immune response. Aim 2: We will determine the efficacy of our delivery system in lipopolysaccharide- challenged mouse models of bacterial sepsis, via imaging and evaluation of anti- inflammatory effects following siRNA-bearing NPSC treatment. The goal of this proposal is to demonstrate the utility of the NPSC platform for immunomodulation. We will build upon the highly efficient cytosolar delivery of siRNA observed in our preliminary NPSC results, evaluating and optimizing their in vivo behavior. These studies will provide critical insights to the translational potential of this vehicle, providing essential preliminary results for applications in specific immune disorders.